Project Summaries for Summer 2009 Session

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Title: Comparison of carotid-femoral pulse vs brachial-ankle pulse wave velocity in children as predictors of cardiovascular risk
Medical Student Research Fellow: Kathryn Bondani
Faculty Preceptors: Bruce S. Alpert, M.D
Grant Somes, Ph.D.

Stiffening of the aorta and large elastic arteries is a sign of vascular aging that has been associated with hypertension, diabetes, and renal failure. Pulse wave velocity (PWV) has been shown to be a useful assessment of arterial stiffness and, therefore, measurement of PWV has become an established predictor of cardiovascular mortality in adults. More recent studies have shown that the process of arterial stiffening begins in childhood with fatty streaks being present even in young children. This atherosclerotic process has been shown to be much easier to reverse in childhood than in adulthood. To date there have been few studies of PWV in pediatric populations. Carotid-femoral PWV (cfPWV) has been considered to be the standard in adults for measuring arterial compliance, but techniques have proven problematic, especially in children. A simpler technique that measures brachial-ankle PWV (baPWV) in children has been reported from our laboratory and others, but its statistical relationship to cfPWV has yet to be reported. Using a simple noninvasive oscillometric technique, the Colin VP-1000, baPWV was measured in 78 subjects [18 African American, 60 Caucasian] ages 11-21yr. The AtCor SphygomoCor, a Doppler pressure tonometer, was used to measure cfPWV in the same subjects. Statistical analyses were performed comparing the two measurements. Preliminary data shows trends of correlation between the two measurements, but further collection and analysis needs to be completed.

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Title: Bone Mineral Density, Inflammatory Markers and Cardiometabolic Risk in a Bi-Racial Cohort
Medical Student Research Fellow: Alex Boucher
Faculty Preceptor: Samuel Dagogo-Jack, M.D., M.Sc., FRC

Type 2 Diabetes Mellitus (T2DM) is known to be associated with alterations in bone mineral density (BMD) and systemic inflammatory tone compared to nondiabetics. It is unknown whether these alterations develop in at-risk individuals before dysglycemia occurs. We, therefore, analyzed a bi-racial cohort of 281 adults (119 Caucasians, 162 African Americans) aged 18-65 years who are normoglycemic offspring of at least one T2DM parent. We determined glycemia did not correlate with BMD and the same held true when divided by race, but we found the cohort had a BMD z-score of 1.15. When divided by race and gender, all of the categories had elevated z-scores. BMC and z-scores showed significant inverse relationships with HbA1c (r=-0.228, p=0.0244 and r=-0.211, p=0.0377, respectively). Peripheral WBC counts showed only a correlation with two-hour blood glucose levels (r=0.185, p=0.0021), yet when divided by race, only African Americans retained significant values (p=0.0174). However, increased leukocyte counts correlated with having three components of the metabolic syndrome (MetS) (p=0.0078). This difference remained when we factored in ethnicity (p=0.0076) with Caucasians having higher leukocyte counts. Our data show that BMD is increased in those at genetic risk for diabetes compared to a reference general population. We also observed that systemic inflammation correlates with accumulation of components of MetS prior to dysglycemia, and long before the development of T2DM.

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Title: Identification of the Cav1.2 α1 N-Terminal Sequence within Human
Smooth Muscle Cells and Its Effects on Cardiovascular Disease
Medical Student Research Fellow: Parker W. Clement
Faculty Preceptor: Jonathan H. Jaggar, Ph.D.

The Cav1.2 family of genes encodes the pore-forming components of calcium-dependent voltage-gated channels within arterial myocytes. Dr. Jaggar's laboratory previously identified a novel Cav1.2 α1 N-terminal splice variant within rat models which when expressed, significantly decreased arterial tone and blood pressure (1). The purpose of this study was to determine if a similar N-terminal splice variant could be isolated from a human aortic smooth muscle cell line by way of Ambion's 5'-RACE PCR protocol. Human aortic smooth muscle cells were purchased from Lonza and cultured. RNA from cultured cells was collected using Invitrogen's TRIzol. 5'-RACE enzymes calf intestine alkaline phosphatase and tobacco pyrophosphatase removed 7-methyl-guanisine caps from collected RNA, and an amplifiable adapter was ligated to RNA for reverse transcription. 5'RACE primers and reverse primers designed for human Cav1.2 α1 exon 2 at base pairs 543 and 365 isolated Cav1.2 α1 exon 1, and PCR products were separated by 1.5% agarose gel. Pertinent bands were excised from the gel, cleaned, and sequenced. From the data collected, exon 1b was isolated from human aortic smooth muscle cells.

To further quantify Cav1.2 α1 N-terminal splice variants, human embryonic kidney cell lines were transfected with exon 1b and exon 1c. Collected protein lysate was used to develop a standard curve through Western Blotting with increasing concentrations of lysates loaded onto sodium dodecyl sulfate resolving gels. Loaded rat arterial protein lysates could then be quantitatively compared to the established curve. Primary antibodies to exon 1b and exon 1c were utilized, as were anti-guinea pig and anti-chicken secondary antibodies respectfully. Results from these experiments have failed to yield expected results; experiments are ongoing.

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Title: Non-Invasive Measurement of Vascular Compliance in Children with Hypertension
Medical Student Research Fellow: Danielle Coury
Faculty Preceptor: Deborah P. Jones, M.D.

Background: Detection of early vascular alterations in hypertensive children could be informative of cardiovascular risk. Pulse wave velocity (PWV) and augmentation index (AI) are non-invasive methods of determining vascular compliance in adults and predict cardiovascular events such as heart attack and stroke. PWV is a measure of arterial distensibility; an increase in PWV corresponds to an increase in arterial stiffness. AI expresses the augmentation pressure (AP) as a ratio to the pulse pressure (PP). AI and AP are also indirect measures of vascular compliance.

Methods: Children ages 10-18 years with hypertension were eligible to participate. PWV was measured using the Colin VP-1000 vascular profiling system (Omron Medical). Aortic systolic and diastolic BP, AI and AP were measured using radial tonometry (SphygmoCor).

Results: Twenty-five subjects have data available for analysis; 14 were male and 18 African-American. Variables are expressed as mean ± SD. Age: 13.8 ± 2.3 years, BMI: 31.3 ± 9.7, height: 167.2 ± 10.9 cm, and weight: 88.3 ± 31.3 kg. Brachial artery (BA) BP was 129.5/77.3 ± 9.2/10.8 mmHg, and aortic (Ao) BP was 111.1/81.0 ± 8.0/10.0 mmHg. PWV was 9.58 ± 1.13 m/s (7.06 - 11.69 m/s); AP 1.75 ± 3.76 mmHg (-5.5-11.5 mmHg); AI was 7.88 ± 10.03 (-11.0-26.0). PWV was significantly correlated with AP (spearman r=0.41, p<0.05) but was not associated with BMI or age. AI was negatively associated with BA pulse pressure (BAPP, r= -0.49, p<0.05) and positively associated with BA DBP (r=0.47, p<0.05). The pulse pressure amplification (BAPP/AoPP) was significantly associated with AP (r = -0.75, p<0.0001) and AI (r = -0.61, p
Conclusions: Non-invasive measurement of vascular compliance was successfully performed in children with HTN. AA race and higher diastolic BP were associated with increased arterial stiffness in hypertensive children.

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Title: Does Therapy with Anti-TNF-α agents Improve Glucose Control and Tolerance in DN2 Patients?
Medical Student Research Fellow: Kyle Fitzgerald Cox
Faculty Preceptor: Solomon S. Solomon, M.D.

Insulin Resistance (IR) is the hallmark of DM2 and may be caused by inflamed fat tissue producing cytokines, notably Tumor Necrosis Factor- Alpha (TNF-α). TNF-α interrupts normal intracellular signaling produced by insulin by phosphorylating the Insulin Receptor Substrate 1 (IRS-1), which ultimately prevents insulin from performing its actions. Consequently, IR ensues and the patient enters an inappropriate prolonged state of hyperglycemia in the presence of increased insulin. We assessed this IR as possible consequence of increased circulating TNF-α via a retrospective chart review on patients who had both an autoimmune disease such as Rheumatoid Arthritis (RA) or Crohn's (CR) and were receiving anti-TNF-α therapy and Type 2 Diabetes.

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Title: Regulation and quantification of retinal transgene products in vivo
Medical Student Research Fellow: Siddharth Desai
Faculty Preceptors: Tonia S. Rex, Ph.D.
Eldon E. Geisert, Ph.D.

Retinal degeneration caused by loss of function mutations can be treated with gene therapy by delivery of the wild-type form of the transgene to the cells of interest. Recent clinical trials have shown success by delivery of a wild-type transgene to the retinal pigment epithelium (RPE) in patients with Lebers Congenital Amaurosis. However successful photoreceptor gene therapy depends on delivery of the correct transgene dose. The photoreceptors are highly metabolically active and delivery of wild-type transgenes to these cells can cause cell death. The goal of this project was to develop a quantitative analytical method for control and determination of transgene product dose in the retina in vivo. Since photoreceptor cells are sensitive to transgene dose, but the RPE is not, we began these proof-of-principle studies in the RPE. Methods: Mice were subretinally injected with a recombinant viral vector carrying enhanced green fluorescent protein (egfp) controlled by an inducible promoter. Five days after injection, the mice received intraperitoneal injections of escalating doses of doxycycline. EGFP fluorescence intensity was measured at 6-7.5 hours post-injection using a Kodak 4000MM imaging station. Results: The peak intensity of EGFP was reached at 6.5-7hrs post-injection. EGFP intensities increased as the dose of doxycycline was increased. Conclusions: Transgene product dose in the RPE can be controlled in a dose-dependent manner in vivo. Further, the alterations in retinal transgene product levels can be measured in vivo. Future studies will test this method in the photoreceptors, and test for equal co-expression of egfp and enhanced yellow fluorescent protein from this bidirectional promoter.

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Title: Defining Novel Mechanisms of the IGF Axis in the Retina Medical Student Research Fellow: Ryan T. Drumright Faculty Preceptor: Robert J. Ferry, MD
Understanding the relationship between the insulin-like growth factor (IGF) axis and apoptosis in retinal cells can help provide some insight into the disease mechanism of diabetic retinopathy. Insulin-like growth factor binding protein-3 (IGFBP-3) has previously been shown to mediate apoptosis in both podocytes and insulin-secreting cells. Human retinal endothelial cells (HRECs) were cultured in high glucose (25 mM) and then treated with recombinant human IGFBP-3. These samples were then analyzed to determine the effect of IGFBP-3 administration on apoptosis as well as phosphorelays in the IGF-I receptor signaling pathway. An ELISA performed for cleaved caspase-3 indicated elevated apoptosis in as little as five minutes after drug administration. Western blotting was used to determine the activity of the IGF-1R signaling pathway. This data is somewhat inconsistent and not complete at the current moment. It does not appear to indicate a decrease in activity of the signaling pathway through IGF-1R and Akt (protein kinase B). This data could indicate that IGFBP-3 caused apoptosis by a mechanism independent of its ability to bind IGF-I and modulate its availability.

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Title: Role of atypical PKC in the Processing and Activation of the Sterol Regulatory
Element-Binding Protein (SREBP-1c), master regulator of lipid synthesis
Medical Student Research Fellow: Shankho S. Ganguli
Faculty Preceptor: Marshal M. Elam, M.D.

It is well known that obesity and persistent hyper insulinemia lead to atherosclerosis induced heart disease, the number one cause of mortality in the western world. The purpose of our study was to determine the most downstream activator of SREBP, the master regulator of lipid synthesis, for the development of antilipemic drug target. Since our lab has previously determined that the insulin-mediated regulation of SREBP occurs via phosphorylation, our goal was to target a specific kinase, PKCζ, in order to determine whether it's the most downstream kinase in the regulatory process of SREBP.

In our experiment, we first treated primary hepatocytes with constitutively active PKC and then measured their intracellular SREBP levels, which were found to be six folds greater compared to control. Using the same protocol, we measured the intracellular SREBP level in the presence of Insulin and inactive kinase PKCζ. Finding a significant reduction in SREBP levels confirmed our hypothesis that phosphorylation of SREBP by PKCζ is necessary for its activation. Next, we carried out autoradiography with 32P labeled ATP with active PKCζ and SREBP. This experiment reaffirmed that SREBP is in fact phosphorylated by PKCζ. In the future, we plan to carry out site directed mutagenesis on potential PKCζ phosphorylation sites on SREBP and repeat the previous experiments to look for differences in results. The purpose of the site directed mutagenesis is to elucidate the role of PKCζ in SREBP regulation and also to determine its exact phosphorylation site in order to facilitate development of drugs that may block the specific amino acid site without interfering with other SREBP sites that are essential for other regulatory.

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Title: Comparative Proteomics of Nuclear Proteins in Spleen Leukocytes from Non-Obese Diabetic (NOD) and Control Strains of Mice
Medical Student Research Fellow: Britni Griffy
Faculty Preceptor: Ivan C. Gerling, Ph.D.

The initiation of the pathologic event leading to destruction of beta cells in Diabetes Mellitus Type 1 has yet to be understood at a molecular level. The aim of this project was to discover spleen leukocyte nuclear proteins that are differentially expressed between Non-Obese Diabetic (NOD) and control strain of mice, Non-Obese Resistant (NOR) and C57BL/6. This detailed look at changes in expression of nuclear proteins and transcription factors may allow for a better understanding of the pathways and mechanisms leading up to the disease and aid in developing new therapies for treatment or prevention. Mice from each of the three strains (n=3 for each group) were sacrificed at 4 weeks of age, a period prior to the onset of autoimmune destruction of beta cells. The spleen leukocyte nuclear proteins were isolated and ran out on 2DE gels. The gels were sequentially stained with a ProQ Diamond phosphoprotein stain and a Krypton total protein stain. A one-way ANOVA analysis was performed to determine proteins of statistical difference in expression levels between the three strains. Heat maps showing hierarchical clustering were generated. We found 704 distinct proteins using the total protein stain, and 583 protein spots when staining for phosphorylation. Analysis by ANOVA identified 83 protein spots using the Krypton stain and 89 using the phosphorylation stain which were differentially expressed between the three strains. Of these, there were 45 proteins of particular interest showing different expression patterns in the NOD mice. The identification of these peptides may provide insight into nuclear proteins and transcription factors that are either activated or muted during the period preceding the development of autoimmune diabetes

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Title: Directed evaluation of candidate autotransporter proteins as vaccine targets for
enterotoxigenic Escherichia coli
Medical Student Research Fellow: Jessica M. Harris
Faculty Preceptor: James M. Fleckenstein, MD

Enterotoxigenic Escherichia coli (ETEC) is a major cause of infection in developing
countries, where it is the most common diarrheal pathogen isolated from children under the age 5 and accounts for hundreds of thousands of deaths annually (WHO, 2009). Vaccine development is a feasible option for prevention of this problem fueled by poor sanitation and lack of a clean drinking water. To identify novel antigens surface expressed during ETEC infection, members of the autotransporter (AT) family were targeted. ATs have two major domains, a highly conserved carboxy terminal outer membrane beta barrel transporter domain and a variable amino terminal passenger domain. Two known member of the autotransporter family, EatA, and TibA, have been described previously in ETEC strain H10407 (Patel et al, 2004;Lindenthal 2001). While TibA is an adhesin, and EatA has serine protease activity, the function of the later AT remains unclear. Two additional AT genes present in ETEC H10407, but absent in commensal strains, were identified using bioinformatics approaches. Passenger domains of these candidates were cloned, expressed, purified, and used for immunoblotting with sera from mice infected with H10407, or convalescent sera from Bangladeshi children (ICDDR,B Dhaka) with acute ETEC diarrhea. Neither preimmune sera from mice or control patient sera [LeBonheur Children's Medical Center] recognized these antigens. Conversely, both immune mouse sera and convalescent sera recognized passenger domains of all 4 autotransporters in H10407. These findings suggest that AT passenger domains are recognized during infection and may be viable candidates for further investigation in vaccine development.

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Title: Effects of P2Y12 Blockade in Thrombus Formation Using in vitro Models
Medical Student Research Fellow: Kourtney Henderson
Faculty Preceptor: Lisa Jennings, Ph. D.

Vessel damage or atherosclerotic plaque rupture results in the exposure of the subendothelial matrix, activation of platelets, amplification of the hemostatic response, and aggregate formation. A number of antiplatelet agents are currently used to prevent aggregate formation in patients with acute coronary syndromes. These treatments are important for the prevention of non-ST elevated acute coronary syndromes and ischemia in percutaneous coronary intervention (6). Furthermore, recent evidence suggests that some antiplatelet agents can disperse formed aggregates (8). The required intravenous administration of GPIIb-IIIa antagonists and the irreversible nature of agents such as aspirin and clopidogrel suggest potential benefits for the use of AZD6140, a reversible, orally administered P2Y12 antagonist currently under evaluation.

Objective: Aggregate stability and dispersion were to be evaluated following addition of AZD6140. Drug-induced thrombus dissolution has previously been observed with the irreversible P2Y12 inhibitor cangrelor (unpublished data, Dr. Jennings' lab), as well as with GPIIb-IIIa antagonists eptifibatide and abciximab (8). Therefore, it is expected that AZD6140 may have similar effects on aggregate dispersion.

Methods: Light transmission aggregometry and microscopic visualization were used to assess the percent platelet disaggregation of freshly formed and aged aggregates following AZD6140 administration.

Results: AZD6140 was found to effectively disperse freshly formed aggregates induced by ADP and collagen. A dose-dependent response was observed, with higher concentrations resulting in more and faster disaggregation. These results suggest that AZD6140 could be effective therapy for patients at risk for ischemic events or those who have recently suffered an ischemic event.

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Title: Analysis of C/EBPβ regulation of the PDK4 promoter
Medical Student Research Fellow: Jae Seung Lee
Faculty Preceptor: Edwards Park, Ph.D.

PDK4 is the most highly regulated of the four pyruvate dehydrogenase kinase (PDK) isoforms. PDK4 is a dedicated kinase in the pyruvate dehydrogenase complex (PDC) which inhibits PDC activity by phosphorylation at three sites. Since PDC catalyzes one of the closely regulated steps in glucose oxidation to acetyl-CoA, phosphorylation by PDK will reduce the cellular glucose oxidation rate. Abundance of PDK4 is controlled by transcriptional mechanisms. Many transcription factors regulate the expression of the PDK4 gene including hepatocyte nuclear factor 4 (HNF4), thyroid hormone receptor (TR), and possibly CCAAT enhancer binding protein β (C/EBPβ). While HNF4 and TR are known to induce expression of PDK4 by binding to adjacent sites in the PDK4 promoter, the role of C/EBPβ has not been investigated. Preliminary data from Dr. Park's laboratory suggests that C/EBPβ stimulates expression of PDK4. We found that there are three regions for C/EBPβ binding between -788 to +78 in PDK4 promoter including -19 to -100, -190 to -290, and -578 to-788. In addition, we investigated the interaction of HNF4 and TR in the induction of PDK4 by thyroid hormone (T3). From the experimental data obtained, we conclude that HNF4 and T3 synergistically induce PDK4 expression. Our data provide additional insight into the molecular mechanisms by which PDK4 gene expression is regulated.

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Title: Screening For Novel And Potent Anti-Cancer Drugs By An Efficient
High-Throughput System Via Anti-HIF-1 Mechanism
Medical Student Research Fellow: Jordan Masters
Faculty Preceptor: Yi Lu, Ph.D.

Cancer is the second leading cause of death in the United States. Nonetheless, current clinical chemotherapeutic agents are far from ideal for cancer treatment. Adaptation to the hypoxic environment seen in solid tumors is critical for tumor cell survival and growth. Hypoxia inducible factor-1 alpha (HIF-1α), a factor that stabilizes in hypoxia and allows cells to survive, has been shown to be elevated in many cancers and high levels are correlated with increased malignancy. HIF-1α is an important transcriptional factor for tumor progression and metastasis, and its activation stimulates a group of HIF-1α-regulated genes, including vascular endothelial growth factor (VEGF), leading tumor cells towards malignant progression. Therefore, a promising approach to cancer treatment is to target HIF-1α. The goal of this project was to develop and validate an Efficient High-Throughput System that has the capability to screen large numbers of potential anti-cancer drugs (small molecule compounds) with anti-HIF-1α activity. MDA-231 cells were cultured and treated with various compounds on a 96-well plate, transfected with a pVEGF-Luciferase plasmid, placed in a hypoxic environment (0.5% O2) for 18 hours, and then assayed for luciferase activity, which reflects HIF-1α activity. Positive compounds were then validated for their anti-cancer and anti-HIF-1α activity via proliferation assays and western blot, respectively. More than 120 compounds were successfully screened for anti-HIF-1α activity via the High-Throughput System. So far we have identified several potentially promising compounds that have shown positive results in the initial anti-HIF-1α screening, anti-cancer growth, and inhibition of HIF-1α protein. These results demonstrate the usefulness and effectiveness of a novel Efficient High-Throughput System for the screening of novel and effective anti-cancer drugs based on the anti-HIF-1α mechanism. Future study will focus on expanding this screening system and promising compounds to other cancer cell lines, and further validation of these promising compounds, initially identified by the High-Throughput System, in the animal model.

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Title: The Role of NF-kB in the Intrinsic Resistance of Renal Cancer Cells to
Interferon's Anti-proliferative Action
Medical Student Research Fellow: Sarah Pierrepont Matthews
Faculty Preceptor: Lawrence M. Pfeffer, Ph.D.

Purpose: The purpose of this experiment was to determine if the presence of the Von Hippel-Lindau (VHL) protein would confer renal cancer cell sensitivity to interferon and/or NF-kB inhibitors.

Experiment Design: Western blots were used to determine the presence of the VHL protein expression and anti-proliferative assays were used to determine the efficacy of Interferon (IFN) and 3 NF-kB inhibitors: BMS-345541, Curcumin, and Bortezomib (Velcade). The renal cell lines were plated with 3000 cells in individual wells of 96 well plates with 3 wells at BMS concentrations of 0, 1, 2, 4, 6, 8 and 10 μM, 3 wells at Curcumin concentrations of 0, 1, 5, 10, 50 and 100 μM, 3 wells at Velcade concentrations of 0, 0.5, 1, 5, 10, 50 and 100 nM, and 3 wells at IFN concentrations of 0, 10, 50, 100, 500, and 1000 U/ml. The renal cell lines were tested for viability using a MTT assay after incubation for 2 days.

Results: Of the renal cell lines with known IFN sensitivity, 5 of the 6 cell lines, including CaKi, SK-RC-02, SK-RC-29, SK-RC-39 and SK-RC-49, had detectable VHL expression in Western blots. Only one IFN-sensitive cell line, SK-RC-17 was VHL negative. Interferon-resistant cell lines SK-RC-04 and SK-RC12 also showed positive VHL expression. IFN-resistant cell lines SK-RC-28 and SK-RC-45 did not express the VHL protein. BMS-345541, Curcumin, and Velcade inhibited the proliferation of the CaKi, SK-RC-04, SK-RC-12, and SK-RC-17 cell lines in a concentration-dependent manner irrespective of VHL presence. While CaKi, SK-RC-04, and SK-RC-12 were VHL positive in Western blot analysis, these cell lines were not significantly more sensitive to any of the NF-kB inhibitors than SK-RC-17, which was VHL negative. It was concluded that IFN treatments were not given at high enough concentrations for a long enough period of time to mirror previously published results of IFN sensitivity.

Conclusion: There was no correlation between VHL expression and sensitivity to NF-kB inhibitors.

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Title: Ocular Formulation of Nutlin-3a for Subconjunctival Delivery in Retinoblastoma Patients
Medical Student Research Fellow: Neal S. Mehta
Faculty Preceptor: Michael A. Dyer, Ph.D.

Retinoblastoma is a severe ocular cancer affecting numerous children each year. Detection and timely treatment often results in survival. It known that human retinoblastomas have an intact p53 gene. The Dyer Group has shown that the p53 pathway is inactivated by a genetic amplification of the MDMX gene. Nutlin-3a, a small molecule inhibitor for the MDM2-p53 interaction, also interacts with MDMX, and could potentially help reestablish the p53 pathway. Currently, the best known solubility of Nutlin-3a was 0.17 mMolar achieved by dissolving Nutlin-3a in Ethanol and subsequently diluting 1:10 in PBS. In order to reduce potential systemic toxicities with current treatment modalities and promote localized therapy, a formulation consistent with FDA published guidelines was developed for a Subconjunctival Delivery of Nutlin-3a. A formulation of 15%/10%/5%/70% (Polypropylene Glycol/Propylene Glycol/Cremophor EL/ PBS) was able to achieve concentrations greater than 25 mMolar with moderate consistency. Proof of concept models will be conducted in the near future to determine the true impact of Nutlin-3a.

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Title: Effects of a High Protein Low Carbohydrate Diet on Various Markers of Glucose and Lipid Metabolism
Medical Student Research Fellow: Lynne Napatalung
Faculty Preceptors: Abbas E. Kitabchi, M.D.
Frankie B. Stentz, Ph.D.

It was been well established that weight reduction improves disease outcomes, but the optimal diet to achieve weight loss and positively influence metabolic markers has yet to be determined. The purpose of our study was to observe the short-term effects of a moderately high protein and low carbohydrate diet in women to assess the diet's effects on weight loss and other metabolic parameters. Specifically, women between the age of 20 and 45 were placed on a diet composed of 40% carbohydrates, 30% protein, and 30% fat over a one-month period. At baseline and one month into the HP-LC diet, various measurements were taken to evaluate the effects of the diet including: weight, waist circumference, body mass index (BMI), blood pressure, fasting blood glucose, 2 hour blood glucose following a mixed meal tolerance test (MMT), triglycerides, and cholesterol. We found that all of the measured metabolic parameters except for 2-hour response to the MMT showed improvement over the course of the one-month period. Response to the MMT actually worsened, a result that may be explained by the limited time period of the study. It should be noted that fasting blood glucose did improve. The subjects will continue on the HP-LC diet for five more months to observe more long-term effects. Additionally, these subjects will be compared to subjects on the traditionally recommended American Diabetic Association (ADA) diet which maintains the same amount of fat, but involves more carbohydrates and less protein.

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Title: A Link between Autoimmunity and Neurodegeneration in Multiple Sclerosis
Medical Student Research Fellow: Michele Roberts
Faculty Preceptor: Michael Levin, M.D.

Objective: To determine if multiple sclerosis (MS) patients develop autoimmunity to neurons that results in neurodegeneration.

Background: Causes of neurodegeneration in MS are largely unknown. Human T-lymphotropic virus type-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) clinically parallels progressive MS and both make antibodies to heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) (Nature Medicine 8, 2002), an mRNA transport protein purified from neurons critical to cell survival. HAM/TSP patients make antibodies to "M9", hnRNP-A1's nuclear translocation sequence (Neuroscience Letters 40, 2006). We tested whether MS patients make antibodies to M9 and if these antibodies cause neurodegeneration.

Design/Methods: IgG was purified from MS patients and tested for immunoreactivity with M9. Anti-M9 and control antibodies were transfected into neurons. Neurons were tested for neurodegeneration with Fluoro-Jade-C. RNA expression was analyzed by microarray. Bioinformatics evaluated functional changes in gene expression. qPCR was used to confirm the microarray and analyze neurons isolated from MS brain. Immunoprecipitation tested whether anti-hnRNP-A1 antibodies interacted with neuronal proteins.

Results: In contrast to controls (n=9), IgG from MS patients (n=35) immunoreacted with M9. Transfection of anti-M9 antibodies into neurons double-labeled with Fluoro-Jade-C, indicative of neurodegeneration. Anti-M9 antibodies significantly altered 866 transcripts (p<0.05, ANOVA) and Gene Ontology classifications showed changes in transcripts related to hnRNP-A1 function; confirming anti-M9 specificity. GeneIndexer, a bioinformatics system that extracts gene function from Medline abstracts, identified a group of genes related to neuron-specific pathways of neurodegeneration. Notably, changes in expression of the spinal paraplegia genes (SPGs) were found, mutations of which cause hereditary spastic paraparesis, a mimic of progressive MS. Changes in gene expression were confirmed by qPCR and were replicated in neurons isolated from MS brain. Immunoprecipitation identified an interaction between hnRNP-A1 and spastin, the encoded protein of SPG4.

Conclusions/Relevance: These findings define a link between autoimmunity, clinical phenotype and neurodegeneration and identified novel therapeutic targets designed to alter the course of progressive MS.

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Title: Role of Phenol Soluble Modulin alpha (PSMα) in the Pathogenesis of Severe Soft Tissue Infections Caused by Strains of Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA)
Medical Student Research Fellow: Scott S. Rosen
Faculty Preceptor: Keith English, MD

Background: Infections caused by community-associated, methicillin-resistant strains of Staphylococcus aureus (CA-MRSA) include life-threatening conditions such as necrotizing fasciitis, sepsis and pneumonia as well as severe soft tissue infections. Identification of specific bacterial components leading to increased virulence in these strains is key to furthering antibiotic research.

Methods: We tested the contribution of the newly identified staphylococcal component PSM-α in a murine model of soft tissue infections.. To do this, we studied an isogenic mutant of one prominent circulating CA-MRSA strain (LAC, USA300) engineered to lack expression of this protein component (LAC/Δ PSM-α). Charles River SKH-1 hairless mice and C57BL6 mice were injected with 108 - 1010 cfu of either LAC or LAC/ΔPSM-α strains of MRSA and observed for lesion size and severity, as well as timing of when lesions began to appear. Additional experiments included treating the infected mice with Daptomycin or Vancomycin (given IP) and observing lesion size/severity - using the above protocol - to further establish the preferred use of Daptomycin in soft tissue infections (based on previous results from Dr. English's laboratory). Results: SKH-1 mice and C57BL6 mice infected with LAC vs LAC/ΔPSM-α developed soft tissue infections of comparable size, but the soft tissue infections of mice infected with strains lacking PSMα did appear to be somewhat less severe and lesions appeared to develop more slowly. Daptomycin treatment of all animals consistently resulted in less severe soft tissue infections (compared with vancomycin treatment).

Conclusions: Our preliminary data indicate that MRSA strains lacking the proposed virulence factor PSM-α cause slightly less severe soft tissue infections.

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Title: Role of VAMP7 in the Cargo Selection of Pre-chylomicron Transport Vesicle
Medical Student Research Fellow: Umair Saleem
Faculty Preceptors: Shadab Siddiqi, Ph. D
Charles Mansbach II, MD

The budding of pre-chylomicron transport vesicle (PCTV) from the intestinal ER is the rate-limiting step in the secretion of chylomicrons from intestinal epithelial cells into the lymphatics. This step represents a potential target for drug therapeutics to control obesity. The primary goal of this project is to determine the role of VAMP7 in the packaging of pre-chylomicrons into PCTV. After properly labeling and isolating rat intestinal ER, several PCTV budding assays were performed. ER that had been incubated with anti-VAMP7 antibodies showed severe attenuation of PCTV generation during PCTV budding assays. Furthermore, titrations of anti-VAMP7 antibodies (2.5 μl, 5.0 μl, 7.5 μl, 10 μl) showed a gradual decrease in PCTV generation with increasing volume of antibody. To test the possibility that VAMP7 antibodies may have blocked PCTV formation by non-specific steric hindrance, a subsequent PCTV budding assay was performed using antibodies of other known proteins associated with ER to Golgi transport (anti-Sec22B, anti-rBet1, and anti-Ykt6 antibodies). Results showed blocking Sec22B, rBet1, and Ykt6 did not have any effect on attenuating PCTV formation. An immunoprecipitation experiment was performed to see the association of VAMP7 with apoB48. ApoB48 is an essential component of chylomicron and has an important role in chylomicron packaging into PCTV. The immunoprecipitation of anti-VAMP7 and anti- apoB48 antibodies in intestinal ER co-immunoprecipitates apoB48 and VAMP7 respectively. VAMP7 plays a role in cargo selection for pre-chylomicrons into their transport vesicles (PCTV) by its association with apoB48. Blocking VAMP7 severely attenuates PCTV budding because it blocks the interaction between VAMP7 and apoB48.

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Title: Effect of Ketone Bodies (β-Hydroxybutyrate and Acetoacetic Acid) on Activation of Human T-lymphocytes
Medical Student Research Preceptor: Kinjal Shah
Faculty Preceptor: Frankie B. Stentz, Ph.D.

Human T-lymphocytes are unique in that in their native state, T-lymphocytes are non-responsive to insulin and do not have insulin receptors. When these T-cells are activated with phytohaemagglutinin (PHA), they express receptors for insulin, growth factors, e.g. IGF-1 and IL-2, and become sensitive to insulin (6). Drs. Stentz and Kitabchi have previously shown that DKA induces in vivo activation of human T-lymphocytes demonstrated by emergence of insulin, IGF-1, and IL-2 receptors (7). DKA is characterized by elevated levels of glucose, ketone bodies, and free fatty acids. This lab has previously shown hyperglycemia-induced activation of human T-cells with de novo emergence of insulin receptors and generation of reactive oxygen species (ROS) and markers of inflammation (8). Similarly, this lab has also shown palmitic acid-induced activation of human T-cells with production of insulin receptors, reactive oxygen species, cytokines and lipid peroxidation (9). However, we have not yet examined the third characteristic of DKA- elevated ketone levels and its ability to activate human T-cells. The goal of this study was to assess the in vitro effect of ketone bodies, such as β-Hydroxybutyrate (β-OH) or acetoacetic acid (AcAt), on activation of human T-lymphocytes at various concentrations and times of incubation for up to 72 hours.

Our preliminary data looked at the effect of ketone bodies on T-cells by looking at markers of oxidative stress by examining levels of ROS using dichlorofluorescein (DCF) assay. From our raw data, we were able to conclude that a 72 hour incubation with β-OH and AcAt does not produce a significant amount of ROS as compared to our positive control of glucose. Similar results were found when the degree of T-cell activation was measured by CD69 receptor expression and the degree of lipid peroxidation determined by malondialdehyde (MDA) using thiobarbituric acid (TBA) assay. Our data indicate that ketone bodies may not play a significant role in the activation of T-lymphocytes as observed in patients in DKA.

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Title: A Novel Mechanism of Dietary Fatty Acid Absorption through the Apical Membrane of Enterocytes
Medical Student Research Fellow: Atur Sheth
Faculty Preceptor: Charles M. Mansbach, M.D.

Fatty acid absorption and transport in intestinal epithelial cells involves transport from the apical membrane of the cell to the cell's endoplasmic reticulum. The currently proposed method of transport involves binding of the absorbed fatty acids (FA) to fatty acid binding protein (FABP) and subsequent diffusion to the endoplasmic reticulum. In this experiment, a potential alternative mechanism of FA transport was studied. This new mechanism was hypothesized to involve vesicular transport of FA from the apical membrane to the endoplasmic reticulum. Vesicles would serve to compartmentalize the absorbed dietary FA so that they would not disrupt intracellular membranes. Vesicles would also provide an efficient mechanism of directing the FA to the endoplasmic reticulum, as compared to a slower, diffusion-mediated movement represented by FABP transport. To determine if vesicles were involved in transport, FA was introduced to rat enterocytes either through their cellular membranes or directly to their cytosol. The samples were then run through a size exclusion chromatography column, and the elutions were compared to protein standard elutions to determine their relative size. The results showed that in samples with FA introduction through the membrane, large structures were eluted from the column; these large structures were absent in the samples where FA was introduced directly to the cytosol. These results show that the FA mode of entry effects structure formation in the cells, implying that vesicles are formed upon FA entry through the membrane. Additional data has confirmed these findings, and research is being done to elucidate further properties of this transport mechanism.

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Title: Pretreatment with Amlodipine to Prevent Myocardial Ca2+ Overloading in Rats with Isoprotenerol-Induced Cardiac Injury
Medical Student Research Fellow: John K. Trussell
Faculty Preceptors: Karl T. Weber, M.D.
Syamal K. Bhattacharya, Ph.D.
Robert A. Ahokas, Ph.D.
Yao Sun, M.D., Ph.D.

Current research on myocardial injury has been examining the effect of hormonal overload from the renin-angiotensin-aldosterone system (RAAS) or by catecholamine neurotransmitters. An RAAS effector hormone or a catecholamine, such as Isoproterenol (Isop), is administered to experimental animals to measure the effects on cardiac function. Isop induces excessive intracellular calcium accumulation (EICA), leading to oxidative stress and myocardial scarring. Zn2+ opposes the action of Ca2+, acting as an antioxidant. Pretreatment with amlodipine (Amlod), a calcium channel blocker, prevents EICA in ALDOST rats. Rats were given Isop (1mg/kg BW) by intraperitoneal injection. The intervention group received three days pretreatment of Amlod (5mg/kg BW/day) by twice-daily gavage. Rats were sacrificed at 8 hours, 24 hours or 7 days, and tissue and blood samples were collected. The total tissue calcium concentrations in all cardiac samples peaked at 8 hours following Isop injection, but declined to near control levels 24 hours and 7 days post-injection. Amlod decreased calcium levels further below control levels at 7 days in most cardiac samples. The total zinc concentrations in all cardiac samples declined at 8 hours and 24 hours, but increased to near control levels at 7 days. Amlod did not significantly alter zinc levels at 7 days. These cation levels as well as 8-Isoprostane assay of the right ventricle indicate that by 7 days, the initial oxidative stress seen at 8 hours has been attenuated. Further collection and analysis of data will allow us to provide a more definitive conclusion as to the cardioprotective effects of Amlod.

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Title: Cardioprotective Effects of ZnSO4 Treatment in Rats with isoproterenol Induced
Myocardial Injury
Medical Student Research Fellow: Sumeet Vaikunth
Faculty Preceptors: Syamal K. Bhattacharya. Ph.D
Yao Sun, M.D., Ph.D.
Robert A. Ahokas, Ph.D.
Karl T. Weber, M.D.

Current research on myocardial injury has been examining the effect of hormonal overload from the renin-angiotensin-aldosterone system (RAAS) or by catecholamine neurotransmitters. An RAAS effector hormone or a catecholamine, such as Isoproterenol (Isop), is administered to experimental animals to measure the effects on cardiac function. Isop induces excessive intracellular calcium accumulation (EICA), leading to oxidative stress and myocardial scarring. Zn2+ opposes the action of Ca2+, acting as an antioxidant. Pretreatment with ZnSO4 enhances endogenous zinc levels. Rats were given Isop (1mg/kg BW) by intraperitoneal injection. The intervention group received three days pretreatment of ZnSO4 (20mg/day) by twice-daily gavage. Rats were sacrificed at 8 hours, 24 hours or 7 days, and tissue and blood samples were collected. The total tissue calcium concentrations in all cardiac samples peaked at 8 hours following Isop injection, but declined to near control levels 24 hours and 7 days post-injection. ZnSO4 decreased calcium levels further below control levels at 7 days in most cardiac samples. The total zinc concentrations in all cardiac samples declined at 8 hours and 24 hours, but increased to near control levels at 7 days. ZnSO4 did not significantly alter zinc levels at 7 days. These cation levels as well as 8-Isoprostane assay of the right ventricle indicate that by 7 days, the initial oxidative stress seen at 8 hours has been attenuated. Further collection and analysis of data will allow us to provide a more definitive conclusion as to the cardioprotective effects of exogenous ZnSO4.

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Title: Animal Model of Regulated Network Study of Glaucoma Related Genes

Medical Student Research Fellow: Allen Vantrease
Faculty Preceptor: Monica Jablonski, M.D.

The anatomical relationship between iris atrophy/pigment dispersion and pigmentary glaucoma is wellknown. Nevertheless, the genetic predecessors of this glaucoma have yet to be fully explored. Using DBA/2J (D2) mice, which are genetically prone to this type of glaucoma due to mutations in Tryp1 and Gpnmb genes, and breeding them extensively with healthy C57BL/6J mice, we were able to generate multiple BXD strains that express the diseases along a wide spectrum. The animal subjects from the 80 different strains are subjected to clinical tests to measure iris degeneration, corneal clouding, and intraocular pressure. Post-mortem histological analysis of the optic nerves and retina are also performed to determine damage to the microanatomy. Because transposition of the mutant alleles alone is not sufficient to generate the increased intraocular pressure characteristic of glaucoma, we suspect that other separate genes play a role in modulating the glaucomatous phenotype. The task of uncovering these novel genes is made possible by our already having sequenced the genomes of the two parental strains. Quantitative trait loci (QTL) analysis may then be used to determine the location of the other regulatory genes that we believe to be modifying the phenotypes in our various mice strains. At this point, a great deal of data must still be obtained. However, some of the results that have already been analyzed suggest that particular genes, unrelated to the Tryp1 and Gpnmb genes, do indeed serve to either increase or decrease susceptibility to glaucoma. We await further data so that similar findings can be said of other features relevant to the pathogenic phenotype.

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Title: A New Multi-Valent Streptococcal Vaccine
Medical Student Research Fellow: Walton, Wiliam J.
Faculty Preceptor: James B. Dale, M.D.

Group A streptococci (GAS), also known as Streptococcus pyogenes causes a variety of illnesses that cover a spectrum ranging from noninvasive streptococcal pharyngitis ("strep throat") to potentially fatal invasive infections like nectrotizing fasciitis and toxic shock syndrome. In addition, some GAS infections may lead to autoimmune complications, namely acute rheumatic fever (ARF), rheumatic heart disease (RHD) or acute poststreptococcal glomerulonephritis (APSGN). Following infection by GAS, the body forms antibodies to M protein epitopes that are protective against infection with the same serotype. Over the course of many years, studies have shown that the protective epitopes of the M proteins are located in the amino-terminal 10-50 amino acids of these surface molecules. These regions are also least likely to give rise to "autoimmune" antibodies that could potentially trigger ARF or APSGN.

Based on this information, a recombinant multivalent vaccine was constructed composed of thirty different M protein amino-terminal peptides located on four different recombinant hybrid proteins. The vaccine was designed to cover 98% of the serotypes causing pharyngitis in North America and 89% and 79% of invasive GAS isolates in the U.S. and Europe, respectively. In addition, the vaccine contains all of the known ARF-causing serotypes of GAS, which could have a significant impact on the global incidence of ARF and RHD. Following immunization of three New Zealand white rabbits via intramuscular administration with 600ug of the vaccine adsorbed to alum at 0, 4, and 8 weeks, immune sera were collected. ELISAs were performed to detect type specific antibodies against the vaccine serotypes of GAS and opsonization assays were performed to detect the percentage of opsonic (protective) antibodies evoked by the vaccine. Using a four-fold increase as a positive threshold for effective immunogencity for the ELISA tests, of the 29 vaccine serotypes tested thus far, one rabbit showed significant immune titers for all M protein serotypes while the other two rabbits developed significant levels of antibodies against 28 of the 29 serotypes tested.

These preliminary data indicate that the new 30-valent streptococcal vaccine is highly immunogenic in rabbits and evokes type-specific, opsonic antibodies that have the potential to provide broad protection against serious and uncomplicated GAS infections.

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Title: The Role of YOR1 and RTA1 in Resistance to Fluconazole in Candida
Medical Student Research Fellow: Wesley G. Willeford
Faculty Preceptor: David P. Rogers, Ph.D.

Candida glabrata is a common cause of mucosal and invasive fungal infections, in large part due to its intrinsic or acquired resistance to azole antifungals such as fluconazole. In C. glabrata clinical isolates, the predominant azole resistance mechanism heretofore elucidated is azole efflux by the ATP-binding cassette (ABC) transporter molecules Cdr1p and Pdh1p which can occur due to increased expression of the genes encoding them. Previous studies have demonstrated that CDR1 and PDH1 gene regulation is controlled by the transcription factor PDR1. In addition, there are RTA1 gene product has been demonstrated to be involved in resistance to 7-aminocholesterol while the YOR1 gene encodes an ABC transporter that has been shown to mediate export of oligomycin. Due to involvement of RTA1 and YOR1 in drug resistance in S. cerevisiae as well as several other genes whose expression is up-regulated in a PDR1-dependent manner in azole-resistant C. glabrata isolates including YOR1 and RTA1 whose orthologues are found in Saccharomyces cerevisiae. The their differential expression in azole-resistant C. glabrata isolates, these genes were candidates for further testing as to their role in azole resistance. We proposed to employ the SAT1-flipper method for disrupting each gene separately in both a wildtype and azole-resistant laboratory C. glabrata strain. During the period in question, SAT1 disruption cassettes were constructed for each gene. We then proposed that fluconazole MIC be measured in the disruptant C. glabrata strains. Such studies prove to be useful in identifying new drug targets necessary to combat Candida infections in the future.

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Title: Role of c-Src in oxidative Stress-Induced Disruption of Tight Junctions in Colon
Adenocarcinoma Cell Monolayers
Medical Student Research Fellow: Courtney Zola
Faculty Preceptor: R. K. Rao, Ph.D.

Epithelial tight junctions that form a fence between the apical and basolateral membranes play a role in maintenance of cell polarity in the epithelial tissues. Uncontrolled expression or activation of c-Src, the protooncogene, plays a role in the carcinogenesis and tumor metastasis in different types of cancers, including colon cancer. Oxidative stress is another crucial factor that promotes cancer growth and metastasis. Previous studies in our laboratory have shown that oxidative stress, induced by hydrogen peroxide, rapidly increased phosphorylation of tight junction proteins and disrupts tight junctions by a Src kinase-dependent mechanism in Caco-2 cell monolayers, an enterocyte cell derived from tumor of colonic origin. In the present study we asked the question whether Src kinase activation and its interaction with the tight junction proteins is involved in this mechanism. Caco-2 cell monolayers were exposed to hydrogen peroxide (20 uM) for 15-180 min. Src activation was measured in live cell by FRET-based Src kinase assay and in cell extracts by immunoblot analysis for c-Src(pY418), the active form of c-Src. Association of c-Src with tight junction protein was determined by co-immunoprecipitation studies. FRET-based Src kinase assay in live Caco-2 cell monolayers demonstrated that hydrogen peroxide rapidly activates c-Src, which was confirmed by a rapid increase in the level of c-Src(pY418). Immunoprecipitation of occludin, a tight junction protein, co-precipitated c-Src, and hydrogen peroxide did not increase the level of such co-immunoprecipitation. However, co-immunoprecipitation of c-Src(pY418) with occludin was rapidly increased by hydrogen peroxide. Pretreatment of cell monolayers with EGF attenuated hydrogen peroxide-induced disruption of tight junctions and tyrosine phosphorylation of occludin, but it did not alter hydrogen peroxide-induced activation of c-Src. These data suggest that hydrogen peroxide activates c-Src associated with tight junctions and induce tyrosine phosphorylation of tight junction proteins. EGF prevents such tyrosine phosphorylation without altering Src activation. Studies suggest that c-Src may directly phosphorylate tight junction proteins and EGF may attenuate tyrosine phosphorylation by modulating other signaling molecules such as protein tyrosine phosphatases.

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Syamal K. Bhattacharya, Ph.D.

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